Ypsilandrosides U-Y, five new steroidal saponins from Ypsilandrathibetica

Phytochemical reinvestigation on the whole plants of Ypsilandra thibetica obtained four new spirostanol glycosides, named ypsilandrosides U-X (1–4), and one new cholestanol glycoside, named ypsilandroside Y (5). Their structures have been established by extensive spectroscopic data and chemical methods. Among them, compound 4 is a rare spirostanol glycoside which possesses a novel 5(6 → 7) abeo-steroidal aglycone, while compound 1 is a first spirostanol bisdesmoside attached to C-3 and C-12, respectively, isolated from the genus Ypsilandra. The induced platelet aggregation activity of the isolates was tested. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00337-0.

Compound 3 was isolated as an amorphous powder and had a molecular formula of C 45 H 72 O 20 as determined by the positive-ion HRESI-MS data (m/z 955.4505 [M + Na] + , calcd. for C 45 H 72 O 20 Na, 955.4509) and 13 C NMR data ( Table 2). Inspection of the NMR spectra (Tables 1 and 2) of 3 revealed that it possessed a spirotanol skeleton with a trisaccharide chain consisting of one rhamnopyranosyl and two glucopyranosyls. Comparing its 1 H and 13 C NMR data (Tables 1 and 2) with those of trillitschonide S6 [17] indicated that they shared the same aglycone. The α-orientations of OH-23 and CH 2 OH-25 were supported by the ROESY correlations between H-23 (δ H 4.00) and H-20 (δ H 3.39)/H-25 (δ H 2.29) (Fig. 3). The absolute configurations and the anomeric configurations of monosaccharides were determined by the same methods with the above compounds. The sequence of the sugar chain at C-3 of the aglycone was established by the HMBC correlations from H-1' (δ H 4.92) to C-3 (δ C 76.8), from H-1'' (δ H 6.31) to C-2' (δ C 77.5), and from H-1''' (δ H 5.04) to C-6' (δ C 69.9) (Fig. 2). Consequently, the structure of 3 was established as (23S,25S)-spirost-5-en-3β,17α,23,27-  13 C NMR data ( Table 2). The UV spectrum of 4 showed absorption maxima at 254.5 nm, suggesting the presence of a conjugated enal system. When comparing its 1 H and 13 C NMR data (Tables 1 and 2) with those of ypsilandroside H [10], it was suggested that they shared the same sugar sequence and the similar aglycone, except for the compound 4 has no hydroxyl substituent at the C-17. The above deduction could be verified by the HMBC correlations from H-21 (δ H 1.13) and H-18 (δ H 0.88) to C-17 (δ C 62.4) and 1 H-1 H COSY correlations between H-16 (δ H 4.59) and H-17 (δ H 1.80) (Fig. 2). The HMBC correlations from H-1' (δ H 5.02) to C-3 (δ C 77.7), from H-1'' (δ H 6.44) to C-2' (δ C 77.9), from H-1''' (δ H 5.82) to C-4' (δ C 77.7), and from H-1'''' (δ H 6.28) to C-4''' (δ C 80.4) confirmed that compound 3 had the same sequence of 3-O-sugar chain as that of ypsilandroside H (Fig. 2). Thus, the structure of 4 was elucidated as (25R)-B-nor(7)-6-carboxaldehyde-spirost-  (Table 2). Its NMR spectra indicated that compound 5 was a cholestane tetraglycosides containing an aromatic ring. Analysis of the 1 H and 13 C NMR data (Tables 1 and 2) of 5 suggested that it was similar to that of parispseudoside A [18], and the major difference was the absence of a glucopyranosyl group at OH-26 site. With the assistance of HSQC experiment, 1 H and 13 C NMR data (Tables 1 and 2)   Because the whole plants of Y. thibetica has been used in folk medicine for treatment of uterine bleeding and traumatic hemorrhage in China, the isolated compounds (1-5) were evaluated for their induced platelet aggregation activity and ADP (adenosine diphosphate) was used as a positive control. Unfortunately, the results showed all isolated saponins did not exhibit the inducing platelet aggregation activity at the tested concentration of 100 μM.

Plant material
The

Extraction and isolation
The dried whole plants of Y. thibetica (110 kg) were crushed and extracted three times with 70% EtOH under reflux for a 3 h, 2 h and 2 h. Then, the combined extract was concentrated under reduced pressure. The crude extract (30 kg) was passed through YWD-3F macroporous resin and eluted successively with H 2 O, 40% EtOH, 75% EtOH, and 95% EtOH, respectively. Evaporated 75% EtOH fraction (crude saponin-rich mixture, 10 kg) was subjected to a silica gel column chromatography (

Ypsilandroside X (4)
Amorphous solid;   Each reaction mixture was directly analyzed by reversed phase HPLC following the above procedure. Each reaction mixture was directly analyzed by analytical HPLC on a Poroshell 120 SB-C18 column (100 × 4.6 mm, 2.7 μm, Agilent) using an elution of CH 3 CN-H 2 O (20:75 → 40:60, v/v) at a flow rate of 0.6 mL/min. As a result, the sugars in the test compounds were identified as d-glucose and l-rhamnose, respectively, by comparing their molecular weight and retention time with the standards (t R 13.90 min for d-glucose; t R 17.72 min for l-rhamnose).

Platelet aggregation assays
Turbidometric measurements of platelet aggregation of the samples were performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation, Havertown,  [19,20]. Rabbit platelet aggregation study was completed within 3.0 h of preparation of platelet-rich plasma (PRP). Immediately after preparation of PRP, 250 μL was incubated in each test tube at 37 °C for 5.0 min and then 2.5 μL of compounds (100 μM) were individually added. The changes in absorbance as a result of platelet aggregation were recorded. The extent of aggregation was estimated by the percentage of maximum increase in light transmittance, with the buffer representing 100% transmittance. ADP (adenosine diphosphate) was used as a positive control with a 59.5 ± 6.1% maximal platelet aggregation rate at a concentration of 10 μM. 1% DMSO was used as a blank control with a 2.7 ± 0.6% maximal platelet aggregation. Data counting and analysis was done on SPSS 16.0, with experimental results expressed as mean ± standard error.

Conclusion
Phytochemical reinvestigation on the whole plants of Y. thibetica obtained four new spirostanol glycosides, named ypsilandrosides U-X (1-4), and one new cholestanol glycoside, named ypsilandroside Y (5). Their structures have been illustrated by extensive spectroscopic data and chemical methods. Among them, compound 4 is a rare spirostanol glycoside which possesses a novel 5(6 → 7) abeo-steroidal aglycone, while compound 1 is a first spirostanol bisdesmoside attached to C-3 and C-12, respectively, obtained from the Ypsilandra species. This investigation enriched the cognition of the chemical constituents in Y. thibetica. Unfortunately, the bioassay results showed the five new saponins have no the activity of inducing platelet aggregation.